1,4,7-Triazacyclononane-Based Chelators for the Complexation of [186Re]Re- and [99mTc]Tc-Tricarbonyl Cores

Metal complexes with the general formula [MI(CO)3(k3-L)]+, where M = Re, 186Re, or 99mTc and L = 1,4,7-triazacyclononane (TACN), NOTA, or NODAGA chelators, have previously been conjugated to peptide-based biological targeting vectors and investigated as potential theranostic radiopharmaceuticals. The promising results demonstrated by these bioconjugate complexes prompted our exploration of other TACN-based chelators for suitability for (radio)labeling with the [M(CO)3]+ core. In this work, we investigated the role of the TACN pendant arms in complexation of the [M(CO)3]+ core through (radio)labeling of TACN chelators bearing acid, ester, mixed acid–ester, or no pendant functional groups. The chelators were synthesized from TACN, characterized, and (radio)labeled with nonradioactive Re-, [186Re]Re-, and [99mTc]Tc-tricarbonyl cores. The nonfunctionalized (3), diacid (4), and monoacid monoester (7 and 8) chelators underwent direct labeling, while the diester (M-5 and M-6) complexes required indirect synthesis from M-4. All six chelators demonstrated stable radiometal coordination. The ester-bearing derivatives, which exhibited more lipophilic character than their acid-bearing counterparts, were prone to ester hydrolysis over time, making them less suitable for radiopharmaceutical development. These studies confirmed that the TACN pendant functional groups were key to efficient labeling with the [M(CO)3]+ core, with ionizable pendant arms favored over nonionizable pendant arms.


Synthetic procedures: 1.1 General
Liquid chromatography electrospray ionization mass spectrometry (LCMS) analyses were performed on a HPLC Gold System (Beckman Coulter, Fullerton, CA).The HPLC was coupled to an ion trap mass spectrometer (LCQ Fleet from Thermo Fisher, Waltham, MA).A Thermo Fisher Scientific BetaBasic C18 column (150 mm x 4.6 mm, 5 μm) was used with a binary linear gradient of 30% to 50% acetonitrile (with 0.1% TFA) in water (with 0.1% TFA) over 30 min (Method 5) and a flow rate of 1 mL/min.LCMS chromatograms were analyzed at the 254 nm wavelength.

Synthesis of N-benzyl-2-bromoacetamide
N-benzyl-2-bromoacetamide was synthesized as previously reported. 1Benzylamine (2.5 g, 0.023 mol) was added to 10 mL of dichloromethane and cooled in an ice bath.Bromoacetyl bromide (2.3 g, 0.011 mol) was added dropwise over 5 minutes, resulting in the immediate formation of a white precipitate.The reaction was allowed to warm to room temperature and stir for 3 h.The reaction mixture was filtered, and the colorless solution was dried under reduced pressure to yield N-benzyl-2-bromoacetamide as a white powder.Isolated yield: 76% (2.0 g).The product was characterized by HRMS, 1

Synthesis of N-benzyl-2-(1,4,7-triazonaneorthoamidyl)acetamide (2)
Compound 1 (165 mg, 1.2 mmol) was dissolved in 5 mL of dry tetrahydrofuran.N-benzyl-2bromoacetamide (300 mg, 1.3 mmol) in 5 mL of dry tetrahydrofuran was added, and the reaction was stirred at room temperature for 18 h.The reaction was filtered to recover the product as a yellow oil, which was then HPLC purified in 10-15 mg batches by semi-preparative HPLC (Method 2, t R = 9.2 min).The HPLC eluate was dried under reduced pressure to yield 2 as a brown oil.Isolated yield: 61% (210 mg).The product was characterized by LCMS, 1  (NEt 4 ) 2 [Re(CO) 3 Br 3 ] was synthesized according to a literature procedure. 3Tetraethylammonium bromide (570 mg, 2.9 mmol) was dissolved in 20 mL of diglyme and heated to 80 °C in an oil bath.Rhenium(I) pentacarbonyl bromide (500 mg, 1.2 mmol) in 20 mL of diglyme was added.The temperature of the reaction was increased to 115 °C and heated for 4 h under a nitrogen atmosphere.During the reaction, a white precipitate formed.The precipitate was filtered and washed with fresh diglyme (10 mL) and cold diethyl ether (10 mL), then slurried in ethanol to remove excess tetraethylammonium bromide and subsequently filtered to give the product, (NEt 4 ) 2 [Re(CO) 3 Br 3 ], as a white powder.Isolated yield: 84% (801 mg).The CO ligand stretching bands were observed by IR spectroscopy at 1847 cm -1 and 1996 cm -1 , matching the values reported in literature. 3

Synthesis of 186 Re/ 99m Tc-labeled complexes
The [ 186 Re][Re(CO) 3 (OH 2 ) 3 ] + precursor was synthesized according to a literature procedure. 4A kit containing borane-ammonia complex (5 mg) was purged with carbon monoxide for 20 minutes.A solution containing [ 186 Re][ReO 4 ] -(74-148 MBq, 2-4 mCi) and 85% phosphoric acid (7 µL) in saline (1 mL) was added to the kit via syringe.The reaction was heated at 65 °C for 15 minutes in a water bath, during which the pressure of the sealed vial was balanced with a 20 mL syringe.After cooling, the kit was opened and the product, [ 186 Re][Re(CO) 3 (OH 2 ) 3 ] + , was analyzed by radio-HPLC (Method 1, t R = 3.1 and 4.8 min).The product presents as two peaks, as reported previously, 5 both of which react with suitable chelators.

IR Spectra
IR spectra were taken on a Thermo Scientific Nicolet Summit Pro FTIR Spectrometer.Wavelengths between 500-4,000 cm -1 were recorded.